PROJECT SUMMARY Post-translational modifications of the histone proteins that package DNA play a central role in regulating transcription and repair of DNA damage. The goal of this research program is to determine the molecular mechanism by which attachment of the small protein, ubiquitin, to histone proteins functions in concert with modifications including methylation and acetylation to regulate gene expression and maintain genome integrity. Since misregulation of histone ubiquitination and methylation characterizes many cancers, the results of these studies can be used to design new drugs that target the molecular machines involved in these pathways. A major focus of this research program will be on the mechanism by which monoubiquitination of histone H2B triggers a cascade of events needed to activate gene transcription, including methylation of histone H3 and recruitment of histone chaperones that assist RNA polymerase in transcribing DNA through a chromatin template. A combination of x-ray crystallography, cryo-electron microscopy and NMR, single molecule biophysics, solution biochemistry and cell-based approaches will be used to study how coactivator complexes such as SAGA bind their chromatin substrates and combine histone deubiquitination, acetylation and recognition of histone methylation to activate transcription. We will also study the mechanism by which H2B ubiquitination triggers histone methylation and determine the distinct mechanism by which different deubiquitinating enzymes mediate nucleosome dynamics. Another aspect of ubiquitin signaling to be addressed in this program is the mechanism by which ubiquitin chains attached to sites of DNA alkylation damage recruit repair enzymes. The molecular insights that will result from the proposed studies will provide a foundation for developing new chemotherapies that target cancers for which there currently are no effective treatments.